Refractive effects of the Gaussian beam on the volume confinement for fluorescence correlation spectroscopy: Experimental and numerical study

The effective detection volume in the case of confocal fluorescence microscopy was investigated in situ by two methods the fluorescence correlation spectroscopy (FCS) of diluted fluorophore solutions, and the measurement of the point spread function (PSF) with calibrated fluorescent microspheres. The determination of the effective volume depends on many parameters and the optical variations in the refractive index were studied experimentally by measuring the PSF under various environmental conditions. The fluorescent microspheres were embedded in different refractive index surroundings media, and excited by a focused Gaussian beam. This allowed us to demonstrate that the mismatches that occur in the refractive index between the media and the sample induce a significant difference in the size of the effective volume and hence in the evolution of the optical resolution. The experimental confocal volume values were compared with those reported in theoretical studies based on the Lanni and Gibson model.